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A kind of stem cell conditioned medium, its preparation method and application

A conditioned medium and stem cell technology, applied in biochemical equipment and methods, culture process, tissue culture, etc., can solve the problems of inactivation of active ingredients, low differentiation efficiency, and difficulty in obtaining materials, and achieve strong differentiation ability and strong cell viability , The effect of taking simple materials

Active Publication Date: 2020-11-17
ZHEJIANG SHENGCHUANG PRECISION MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its composition is complex, and the post-processing is complicated, and 35°C water bath and ultrasound are used in the process of preparing liposome freeze-dried powder, which can easily lead to the inactivation of active ingredients in human umbilical cord MSC serum-free culture medium
[0010] It can be seen from the above that the stem cell conditioned medium in the prior art is mostly derived from bone marrow, umbilical cord placenta, adipose stem cells, etc., and there are problems such as difficulty in obtaining materials, shortage of sources, and low differentiation efficiency.

Method used

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  • A kind of stem cell conditioned medium, its preparation method and application
  • A kind of stem cell conditioned medium, its preparation method and application
  • A kind of stem cell conditioned medium, its preparation method and application

Examples

Experimental program
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Embodiment 1

[0042] Example 1 Preparation of uterine blood stem cells

[0043] 1. Cell preparation process

[0044] Young women aged 25-35 without gynecological infectious diseases use menstrual cups or tampons to collect menstrual blood (about 8-10mL). Transfer to the menstrual blood collection fluid, within 24-48 hours after the sample is obtained, and before being sent to the processing laboratory, it should be transported or stored at 4°C. Filtrate with a 70-micron filter in a biological safety cabinet, collect the filtrate, and centrifuge the filtered whole blood at 2800rpm, liters 5, speed down 1, and 10min, take 5ml of the supernatant for pathogen detection, and discard the rest of the supernatant for blood cell precipitation Resuspend in a certain volume of PBS (Phosphate Buffer Saline, phosphate-buffered saline) (usually blood cell sediment volume: PBS volume = 1:1-1:5), and carefully and gently inject the PBS and menstrual blood mixture with a pipette In the human peripheral bl...

Embodiment 2

[0056] Conditioned medium preparation of embodiment 2 uterine blood stem cells

[0057] Take P5 uterine blood stem cells that are in good condition, pass the immunophenotype test, and have strong three-line differentiation ability, and use 1×10 6 cells / bottle inoculated to T 175 Cell culture flask, when the cell confluency reaches 80%, replace with fresh serum-free DMEM / F-12 medium, 20ml / bottle, continue culturing for 24 hours, collect the supernatant, remove dead cells by centrifugation, and collect the filtrate by filtration through a 0.22 μm filter membrane. Store in a -80°C refrigerator after aliquoting or make freeze-dried powder.

Embodiment 3

[0058] Embodiment 3 human fibroblasts are cultured

[0059] MRC-5, human embryonic lung fibroblasts were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. When the cell confluency reached more than 80%, it was digested with 0.25% trypsin for about 1min and observed under the microscope. The digestion time was within 5min. After 70% of the cells fell off, add 10% FBS MEM medium to stop the digestion, gently blow the wall of the bottle to minimize cell loss, collect the cell suspension, centrifuge at 300g for 5 minutes, discard the supernatant, add an appropriate volume of MEM medium containing 10% FBS to resuspend, and carry out passage Amplify, cryopreserve (the ratio of cryopreservation solution MEM:FBS:DMSO=7:2:1) or use in experiments. The ratio of medium is MEM:FBS=9:1.

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Abstract

The invention relates to a stem cell conditioned culture medium as well as a preparation method and application thereof. Specifically, the stem cell conditioned culture medium is prepared through thefollowing method: (1) obtaining uterine blood mesenchymal stem cells from menstrual blood; (2) culturing the uterine blood mesenchymal stem cells in a culture medium without antibiotics and passagingto P5 to P10 generations; (3) culturing the P5 to P10 generation uterine blood mesenchymal stem cells at an exponential phase in a serum-free culture medium without the antibiotics and activating andculturing for 24 to 48h under a serum-free starvation condition; (4) taking supernatant and filtering to remove the uterine blood mesenchymal stem cells to obtain filtrate, i.e., the stem cell conditioned culture medium. The method provided by the invention is simple and exogenous substances are not introduced; side effects including allergy and the like can be reduced. Furthermore, the conditioned culture medium has very good in-vitro and in-vivo anti-oxidization and anti-ageing effects.

Description

technical field [0001] The invention relates to a stem cell conditioned medium, its preparation method and application. It belongs to the field of stem cell anti-oxidative aging. Background technique [0002] Skin refers to the tissue on the surface of the body that covers the muscles. It is the largest organ in the human body and mainly undertakes functions such as protecting the body, perspiration, and feeling cold, heat, and pressure. The skin covers the whole body, and it protects various tissues and organs in the body from physical, mechanical, chemical and pathogenic microorganisms. With the proliferation of age and the influence of harmful factors in the environment, a series of problems such as aging, wrinkles, dullness, allergies, dryness, and red blood will appear on the skin. The main manifestations of skin aging are dry skin, weakened elasticity, slowed epidermal renewal, decreased keratinocyte vitality, and weakened repair ability of the epidermis after damage...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775A61K35/28A61P17/18A61K8/99A61Q19/08
CPCA61K8/99A61K35/28A61P17/18A61Q19/08C12N5/0665C12N2500/90
Inventor 朱丽萍陈露郭垚示项春生
Owner ZHEJIANG SHENGCHUANG PRECISION MEDICAL TECH CO LTD
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